Actin/Myosin-V- and Activity-Dependent Inter-synaptic Vesicle Exchange in Central Neurons  Michael W. Gramlich, Vitaly A. Klyachko  Cell Reports  Volume 18, Issue 9, Pages 2096-2104 (February 2017) DOI: 10.1016/j.celrep.2017.02.010 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 18, 2096-2104DOI: (10.1016/j.celrep.2017.02.010) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Actin and Myosin V Support Vesicle Motility during ISVE (A–C) Example vesicle tracks that leave their respective synapse of origin and travel along the axon for CT (A), Myo-1 (B), and Lat-A (C) conditions overlaid on a corresponding whole-synapse image, with synapses outlined (dashed outlines). Frame-to-frame velocity is represented by the color bar. (D) Cumulative distributions of frame-to-frame angular displacement outside synapses for CT (black), Myo-1 (red), and Lat-A (yellow). Dashed line indicates a purely diffusive motility. Solid lines are computational models of the data (Supplemental Experimental Procedures Appendix 2). (E) Same as (D) for angular displacement inside synapses. (F) Cumulative distributions of instantaneous velocity outside synapses. Solid lines are analytical fits to the data (Supplemental Experimental Procedures Appendix 2). (G) Same as (F) for inside synapses. (H) Frequency of pausing outside the synapses measured as a function of distance because vesicles spend little time (∼2–5 s) along an axon but travel a significant distance (>1,000 nm). (I) Frequency of pausing inside the synapse measured as a function of time because vesicles spend most of time inside a synapse (∼20–40 s) but do not travel large distances (∼300–400 nm). The scale bar in (C) is 1 μm and is the same for (A)–(C). In (H) and (I), error bars represent means ± SEM. In all figures, ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05. Cell Reports 2017 18, 2096-2104DOI: (10.1016/j.celrep.2017.02.010) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Myosin V Underlies Fastest Component of Directed Vesicle Motility (A and B) Representative examples of CT (A) and Myo-1 (B) tracks exhibiting fast-directed runs (magenta), intermediate motility (green), and pauses (blue), as determined by a transient motion analysis (Supplemental Experimental Procedures Appendix 1). Tick marks are 500 nm. (C) The maximum displacement of vesicles that engage in at least one fast-directed run. (D) Same as (C) for the average length of individual fast-directed runs. (E) Same as (C) for vesicle velocity per run. (F) Same as (C) for the time spent in fast-directed runs. Cell Reports 2017 18, 2096-2104DOI: (10.1016/j.celrep.2017.02.010) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Actin Mediates Myosin V Function in Long-Range Vesicle Motility (A) The maximum vesicle displacement during observation in different conditions. (B) The average length of a fast-directed run. (C) The average velocity during a directed run. (D) The average duration of a fast-directed run. Number of tracks that had at least one fast directed run: CT: 115; Myo-1: 31; Lat-A: 48; PBP: 29; DMSO: 66; Lat-A/Myo-1: 43. All data are presented as mean from cumulative distribution fits ± SEM to make differences clearly distinguishable. Cell Reports 2017 18, 2096-2104DOI: (10.1016/j.celrep.2017.02.010) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Vesicle Exit from the Synapse and Fast Long-Range Motility Are Activity Dependent (A) A cartoon model showing labeled vesicles exiting from the synapse at a constant rate (ζ) that are replaced by un-labeled vesicles at the same rate (χ). Over time, reduction in labeled vesicles inside the synapse reduces the apparent vesicle exit rate (ζobs). (B) The fraction of labeled vesicles remaining in the synapse over time. Stimulus duration is marked by dashed lines. CT-NoStim, CT with no stimulation during the entire 0- to 40-s period; CT-Stim and Myo-1-Stim, CT or Myo-1 with stimulation during 10- to 20-s period. Lines are analytical (solid line) and computational (dashed lines) models. (C) The slope from (B), with SE, for each condition plotted as a function of specific time period. (D) The maximum displacement of vesicles in the absence (gray) or presence (black) of stimulation for a subset of vesicles that travel beyond the synapse (600 nm) and have at least one fast-directed run, plotted as cumulative fraction. (E) Same as (D) for the length of individual fast-directed runs. (F) Same as (D) for the velocity of fast-directed runs. (G) Same as (D) for the time spent in a fast-directed run. Cell Reports 2017 18, 2096-2104DOI: (10.1016/j.celrep.2017.02.010) Copyright © 2017 The Author(s) Terms and Conditions